A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.
A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.
复制标题
DOI：
10.1371/journal.pone.0141074
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Lee DA
中科院分区：
文献类型：
--
作者：
Somanchi SS;McCulley KJ;Somanchi A;Chan LL;Lee DA
自然杀伤细胞（Natural killer， NK）属于免疫系统的天然手臂，活化后的NK细胞虽然可以通过分泌细胞因子来调节免疫应答，但其主要的效应功能是通过裂解靶细胞。因此，细胞毒性试验是研究NK细胞功能的核心。51Chromium释放试验是细胞毒性试验的“金标准”，然而，由于担心与放射性化合物的使用和处置相关的毒性，因此对非放射性方法有很大的兴趣。我们以前使用钙黄蛋白释放试验作为研究NK细胞毒性的非放射性替代方法。在这项研究中，我们发现钙黄蛋白释放测定在不同肿瘤靶点的动态范围内是不同的，并且被捕获的钙黄蛋白可能在裂解的肿瘤靶点的凋亡小体中保持未释放或不完全释放，从而导致特异性裂解百分比的低估。为了克服这些限制，我们使用Cellometer视觉图像细胞仪开发了一种新的细胞毒性测定方法，并将该方法与用于测量NK细胞毒性的标准钙黄蛋白释放法进行了比较。使用肿瘤细胞系K562、721.221和Jurkat，我们在这里证明，在相同的实验设置下，与标准钙黄蛋白释放试验相比，图像细胞术显示靶细胞的特异性裂解率显着提高。图像细胞术能够通过从活靶细胞计数中排除较暗的细胞和较小的凋亡小体来准确地分析活靶细胞。基于图像细胞术的细胞毒性检测是一种简单、直接和敏感的方法，是常规细胞毒性检测的一种有吸引力的选择。
Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the “gold standard” for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.